Use of an adsorbent gel for eliminating and purifying biomolecules

ABSTRACT

The invention concerns the use of an adsorptive size exclusion chromatography gel, said gel essentially consisting of a polysaccharide matrix whereon is grafted a polymer coupled with an affinity ligand and having a cleavage threshold ranging between 2 kDa and 60 kDa for eliminating a purifying biomolecules.

The present invention relates to the use of an adsorbent gel combiningthe properties of size exclusion and affinity chromatographies (AdSEC,for “Adsorptive Size Exclusion Chromatography”).

The principle of an AdSEC gel results from the fusion of twochromatographic techniques: size exclusion and affinity, so as to obtainsupports combining the most advantageous properties thereof.

Size exclusion chromatography (gel filtration) allows the separation ofmolecules according to their steric bulk alone during their passivediffusion in a molecular sieve (gel). The largest molecules cannotpenetrate the crosslinked matrix and are consequently excluded morerapidly from the column. This technique possesses the characteristicfeature of not exhibiting interactions between the support and themolecules, and therefore of being relatively only slightly sensitive tothe biochemical conditions (pH, ionic strength) of the solution. On theother hand, because of its principle of diffusion, the limiting factorsfor its use are generally a long operation time (because low flow ratesare used), as well as a relatively limited deposition of samples (1 to5% of the column volume).

Affinity chromatography is based on molecular interactions between thesupport (matrix onto which affinity ligands are grafted) and themolecules to be separated. Among these affinity ligands, immobilizedmetal ions, introduced in 1975 by Porath et al. (Nature, 1975, 258,598-599), represent a method of separation based on the interactions(coordination bonds) between biomolecules in solution and metal ionsimmobilized on a support; Zn(II), Cu(II), Ni(II) and Co(II) ions are themost commonly used. This is described as immobilized metal ion affinitychromatography (IMAC).

The combined use of the principles of size exclusion and affinitychromatographies (AdSEC) has been discussed by Porath et al. (Int. J. ofBio-Chromatogr., 1997, 3, 9-17). These authors have shown thatiminodiacetic derivatives of dextran bearing metal ions as affinityligand allow size exclusion and are capable of effectively concentratingsolutions by their properties of adsorption and affinity. These authorshave shown that an AdSEC gel column having a volume of 5 ml could bind ahigh percentage of compounds having a molecular weight of between 5 kDaand 50 kDa and concentrate them about 1000 fold in a single operation.

Such supports make it possible to adsorb the smallest molecules (havingaffinity for the grafted ligand) at high rates and volumes (notpermitted in gel filtration). Moreover, during the synthesis of theadsorbent gel, the threshold of accessibility to the affinity ligand maybe modulated during the synthesis of the gel according to the size ofthe biomolecule to be removed or to be purified.

Terminal renal insufficiency currently affects 22,000 people in Franceof which 20,000 are treated by iterative hemodialysis. Only 1800 canhope to undergo transplants each year, knowing that a quarter of themwill return within 5 years to hemodialysis because of a rejection whilewaiting for a new transplant.

The survival of the uremic individual, all methods considered, canexceed 25 years if they do not suffer from a severe cardiovascularcondition. In this case, the quality of survival is profoundly impairedover the years by the osteoarticular complications of terminal uremia,at the forefront of which there are described erosive arthropathiessubsequent to depositions of β2-microglobulin (β2-M).

The mechanism of onset of these arthropathies begins as soon as therenal insufficiency responsible for accumulation of β2-microglobulinappears. This protein, having a molecular weight of 11,800 Da, willaccumulate in the body over the years and become selectively depositedat the level of the cervical disks, of the shoulders, of the hips and ofthe wrists. Cardiac and digestive depositions have been reported. Thesedepositions will make fragile the joint and the adjacent bone up tototal destruction of the joint. Thus, a breakdown of the vertebralbodies is observed which can cause medullary compression with loss ofcontrol of the four members, irreversible articular luxations, loss ofprehension in the hands and pseudofractures of the hip. Ductal nervecompressions are observed such as the carpal tunnel syndrome.

These complications irremediably lead the uremic individual towardinvalidity and the bedridden state which conventional methods ofdialysis cannot prevent. A transplant allows these lesions to bestabilized.

To effectively prevent these complications, it is important to be ableto effectively purify the polluting components of blood, in particularβ2-microglobulin, which are synthesized daily by the body and which arenot, or not sufficiently, removed by the defective kidneys in dialyzedpatients.

The purification of these various biomolecules can only be done onartificial membranes during dialysis, which are currently notsufficiently effective in spite of purification by filtration andnonspecific membrane adsorption.

The existing techniques for removing biomolecules, includingβ2-microglobulin, are currently of 3 types:

1. Removal of Biomolecules by Hemodialysis

Hemodialysis is a technique intended for subjects suffering from partialor complete renal insufficiency (FIG. 1). It consists in extracorporealtreatment of blood, providing the same functions as the kidney using amembrane process. The essential part of the hemodialyzer (1) is anexchange membrane, on either side of which circulate countercurrentwisethe patient's blood and the dialyzate obtained from the hemodialysisgenerator (2). This technique allows the purification of the smallmolecular weight compounds polluting the blood, such as urea, aminoacids, inorganic salts, which are normally removed by the kidney. In thecase of serum β2-microglobulin, the various dialysis membranes commonlyused possess two antagonistic properties:

capture of β2-microglobulin by nonspecific adsorption on the membrane,

generation of β2-microglobulin by detachment of this molecule which isnoncovalently associated with the surface of nucleated blood cells inthe major histocompatibility complex type I.

The degree of generation of β2-microglobulin is one of the criteriawhich define the biocompatibility of the membranes. Thus, endowed withthese two antagonist properties, some membranes lead overall, during ahemodialysis session, to an increase in the concentration ofβ2-microglobulin, whereas others reduce it.

However, regardless of the membranes used, these results level out overperiods of over one year. Thus, it has been observed that the plasmalevel of β2-microglobulin in uremic patients after fifteen months ofdialysis was invariably increased to be between 40 and 50 mg/l (against1 to 2 mg/l in healthy patients). Such problems of biocompatibility alsoexist for the other biomolecules.

2. Removal of the Biomolocules by Hemofiltration

Once per month, the dialyzed individual is subjected to anultrafiltration session. The module used (1) possesses a higher cut-offthan in hemodialysis (average cut-off of 40 kDa) and allows the removal,by filtration, of the small molecules from plasma, including thesmallest proteins, such as β2-microglobulin (FIG. 2). During anultrafiltration session, the loss of plasma water is compensated by anequivalent supply of physiological saline (3).

The qualitative results, with respect to the removal of β2-microglobulin(purification and generation of this molecule by ultrafiltrationmembranes), are similar to those obtained in hemodialysis. There is thusa great influence of the nature of the membrane and of the duration ofthe hemofiltration. While some membranes appear to remove moreβ2-microglobulin over 5 hours (one session), a leveling out of theresults is also observed over time. At the quantitative level, itappears that about 50% of the serum β2-microglobulin is removed perhemofiltration session. However, even if this technique is moreeffective for the purification of β2-microglobulin than hemodialysis, itremains inadequate for preventing and stopping the appearance of thedisease. Furthermore, this technique has the disadvantage of removingnumerous other small proteins apart from β2-microglobulin, since theultrafiltrate is removed permanently.

3. Column/hemodialyzer Coupling

This method has been presented as an alternative to the customaryhemodialysis and ultrafiltration methods (Nakazawa et al., Int. J.Artif. Organs, 1994, 17, 203-208). It consists in a serial adsorption ofthe biomolecules on a porous cellulose gel (350 ml of adsorbent),followed by conventional hemodialysis. In the case of β2-microglobulin,the gel is described as having a theoretical capacity forβ2-microglobulin of 1 mg per ml of adsorbent. The results obtained arethe best described in the literature, since in a patient in whom theinitial β2-microglobulin level was 30 mg/l, this system made it possibleto reduce the β2-microglobulin concentration to 10 mg/l final after 6months of treatment. The authors presented an improvement in delayingthe appearance of amyloid deposits in 2 cases out of 3, in theirpatients after therapy.

However, a drop in the concentration of some serum molecules (retinolbinding protein, lysozymes) is also observed after treatment. Thisphenomenon is attributable to the direct passage of the blood throughthe adsorbent, which is likely to cause problems of biocompatibility.

Thus, the existing techniques for removing β2-microglobulin and otherbiomolecules have mainly two limits:

the biocompatibility of the supports, in particular for the generationof β2-microglobulin, that is to say the equilibrium between nonspecificadsorption on the membrane and the generation of β2-microglobulin duringthe passage of the cells in contact with them; this equilibriumdetermines the quantity of β2-microglobulin really removed during ahemodialysis or hemofiltration session.

the specificity of the substrate: indeed, the techniques ofhemofiltration and of a specific binding with ligands coupled to gelslead to the undesirable removal of other molecules from serum.

A device for removing β2-microglobulin or any other biomolecule shouldtherefore combine satisfactory (quantitative) removal with specific(qualitative) removal of the molecule in question.

In the present invention, the inventors therefore set themselves asobjective:

the use, in a device intended to remove biomolecules, of an adsorbentgel combining the properties of size exclusion and affinitychromatographies, said gel essentially consisting of a polysaccharidematrix onto which is grafted a polymer coupled to an affinity ligand(AdSEC, for “Adsorptive Size Exclusion Chromatography” gel) and havingan adjustable cut-off of between 2 kDa and 60 kDa,

the use of an AdSEC gel for separating and purifying biomolecules havinga molecular weight of between 2 kDa and 60 kDa,

a device intended for the removal of biomolecules having a molecularweight of between 2 kDa and 60 kDa comprising an ultrafiltration moduleoptionally upstream and in series with a dialysis module and using anAdSEC gel column having an adjustable cut-off of between 2 kDa and 60kDa, said column being mounted branching off from said ultrafiltrationmodule; this device makes it possible to dispense with the problems ofbiocompatibility and to specifically remove the desired biomolecules,

a device for purifying biomolecules having a weight of between 2 kDa and60 kDa using an AdSEC gel column having an adjustable cut-off of between2 kDa and 60 kDa, said column optionally branching off from a filtrationsystem; this device makes it possible to separate normal biomoleculesand biomolecules modified for example by glycation.

In one advantageous embodiment, the polysaccharide matrix is agarose oris based on an agarose derivative, the polymer may be polyethyleneglycol (PEG) or polypropylene glycol (PPG) and the affinity ligand maybe, for example, a metal-chelating agent coupled to metal ions, aprotein, a peptide, an enzyme substrate or an enzyme inhibitor.

In a preferred embodiment, the adsorbent gel consists of a matrix basedon an agarose derivative onto which is grafted polyethylene glycolcoupled to iminodiacetic acid (IDA) itself coupled to metal ions, forexample copper(I) ions; this complex is called IMAdSEC (“ImmobilizedMetal ion Adsorptive Size Exclusion Chromatography”) gel.

In an also preferred embodiment, the cut-off of the adsorbent gel is 20kDa, thus allowing the removal or the purification of biomolecules whosemolecular weight is less than 20 kDa, in particular serumβ2-microglobulin.

The purification system according to the present invention possesses thecharacteristic feature of placing the adsorbent gel for the biomoleculeto be removed branching from the circulation system for purifying. Thus,when blood is purified, there is at no time contact between the gel andthe formed elements of the blood, therefore the problems ofbiocompatibility (for example generation of β2-microglobulin throughcontact with the nucleated cells of the blood) or hemolysis of the cellsin contact with the gel are avoided.

Furthermore, unlike the other techniques currently used, thepurification of the biomolecule to be removed or to be purified iscarried out using a ligand which will retain only this molecule. Thisspecificity is obtained by virtue of the double sieving of theultrafiltration membrane (which retains for example the formed elementsof the blood and the large serum molecules) and of the AdSEC gel whichprevents access to the ligand for other molecules with affinity for theaffinity ligand but whose size is greater than the cut-off of the gel.

The other advantage of the use of this AdSEC gel is its ease ofregeneration. For example, when a metal is used as affinity ligand, itmay be chelated by a solution of EDTA, which makes it possible to detachany molecule adsorbed onto the gel, thus allowing cleaning of the gel,its regeneration with a new metal load and its sterilization.

The removal system according to the invention may be used for example inthe context of kidney dialysis; in this case, there is an additionaladvantage linked to the fact that the fraction purified by passage overthe AdSEC gel returns to the patient, thus limiting losses of otherelements present in the blood.

In addition to the preceding features, the invention further comprisesother features which will emerge from the description which follows,which refers to examples as well as to the appended figures in which:

FIG. 1 represents the general diagram for renal dialysis; (1)hemodialyzer, (2) hemodialysis generator, (3) pump,

FIG. 2 represents the diagram for a hemofiltration by ultrafiltration;(1) hemofilter, (2) hemodialysis generator, (3) physiological saline,(4) pump,

FIG. 3 illustrates the chromatographies on metal ions (copper)immobilized on 3 types of gels: A Sépharose® 4B-IDA-copper, peak 1:nonadsorbed proteins; peak 2: elution at pH 6.0; peak 3: elution at pH5.0; peak 4: elution at 4.0; peak 5: elution at pH 3.0; peak 6: 25 mMEDTA. B Novarose®-IDA-copper, peak 1: nonadsorbed proteins; peak 2:elution at pH 6.0; peak 3: elution at pH 5.0; peak 4: elution at pH 4.0;peak 5: elution at pH 3.0; peak 6: 25 mM EDTA. CNovarose®-PEG/IDA-copper (IMAdSEC), peak 1: nonadsorbed proteins; peak2: elution at pH 6.0; peak 3: elution at pH 5.0; peak 4: elution at pH4.0; peak 5: elution at pH 3.0 (1st peak); peak 5′ elution at pH 3.0(2nd peak); peak 6: 25 mM EDTA,

FIG. 4 illustrates the electrophoretic analysis of the fractionsseparated by chromatography illustrated in FIG. 3; the numberscorrespond to the fractions separated by chromatography in FIG. 3; ASépharose® 4B-IDA-copper. B Novarose®-IDA-copper. CNovarose®-PEG/IDA-copper (IMAdSEC). This figure illustrates thespecificity of the IMAdSEC gel for β2-microglobulin relative to the twoother types of gel,

FIG. 5 illustrates the analysis by mass spectrometry of the proteincomposition of the starting ultrafiltrate and of the fraction retainedon IMAdSEC gel. A (I) spectrum for the ultrafiltrate, (II) deconvolutionof the spectrum (a) calculation of the mass of β2-microglobulin (b)calculation of the mass of albumin. B (I) spectrum for the purifiedfraction, (II) deconvolution of the spectrum and calculation of the massof β2-microglobulin,

FIG. 6 illustrates the capacity of the IMAdSEC gel for β2-microglobulin,

FIG. 7 illustrates the mounting, on a branch, of a filtration module ofthe purification device according to the invention; (1) ultrafiltrationmodule, (2) column containing the IMAdSEC gel, (3) pumps, (4)ultrafiltrate,

FIG. 8 illustrates the capacity of the device illustrated in FIG. 7 forthe removal of β2-microglobulin from an ultrafiltrate of a uremicpatient,

FIG. 9 illustrates the electrophoretic analysis of the fractionsseparated by chromatography illustrated in FIG. 8; 1: ultrafiltrate; 2:15 minutes of passage over the IMAdSEC gel; 3: 30 minutes of passageover the IMAdSEC gel; 4: 120 minutes of passage over the IMAdSEC gel; 5:fraction eluted at pH 5.0; 6: fraction eluted at pH 4.0; 7 fractioneluted at pH 3.0; 8: fraction eluted with EDTA, 9: protein standard,

FIG. 10 represents a hemodialysis system comprising the device accordingto the invention; (1) hemofilter, (2) hemodialyzer, (3) IMAdSEC column,(4) hemodialysis generator, (5) blood pump and (6) ultrafiltration pump.

EXAMPLE 1 Determination of the Specificity and of the Capacity of anIMAdSEC Gel: (Novarose®-PEG/IDA-copper) for β2-microglobulin

1. Synthesis of the Novarose®-PEG/IDA-copper Gel:

Step 1: coupling of PEG and creation of the cut-off of the gel:

10 g of Novarose® Act High 100/40 (INOVATA, Bromma, Sweden), previouslydried by suction, are taken up in 5 ml of 1 M Na₂CO₃, pH>12 and 5 ml ofdeionized water. 5 ml of 1 M Na₂CO₃, pH>12, 5 ml of deionized water and30 ml of NH₂-PEG-NH₂ at 10% in 1 M Na₂CO₃, pH>12, are added. The mixtureis left under gentle stirring at room temperature (22° C.) for 1 to 24hours depending on the desired cut-off (this time is 4 hours for acut-off of 20 kDa which is the desired cut-off for β2-microglobulin).

Step 2: coupling of the ligand:iminodiacetic acid (IDA).

The gel obtained in step 1 is rinsed on sintered material (by suction)with a solution of deionized water. It is resuspended in a solutioncomprising 15 ml of 1 M Na₂CO₃, pH>12, 15 ml of deionized water, and 10ml of a solution of IDA at 10% in 1 M Na₂CO₃, pH>12. The mixture is leftunder gentle stirring at room temperature (22° C.) for 48 hours. TheIMAdSEC gel is rinsed on sintered material successively with deionizedwater, with a 1 M solution of sodium hydroxide, with deionized water,with a 0.1M solution of hydrochloric acid, and then with deionizedwater. The gel thus obtained is kept at 4° C. in a solution of 20%ethanol until it is used.

Step 3: coupling of the metal ions (copper Cu II ions):

The metal load is prepared using an aqueous solution of copper sulfateat 50 mM under conventional conditions.

2. Preparation of the Biological Solutions

The products are derived from the hemofiltration of blood during anultrafiltration session in the context of the treatment of uremicpatients (FIG. 2). Ultrafiltrates (pH 7.2, 13 mS/cm) are used whoseβ2-microglobulin concentration varies from 7 to 20 mg/l according to thepatients.

3. Specificity of the Novarose®-PEG/IDA-copper Gel for β2-microglobulinCompared with Gels without Sieving Sépharose® 4B-IDA-copper andNovarose®-IDA-copper

Procedure:

3 gels were tested: Sépharose® 4B-IDA-copper, Novarose®-IDA-copper (IMACgels), and Novarose®-PEG/IDA-copper (IMAdSEC gel), for their capacity toadsorb the molecules of the ultrafiltrate from a uremic patient. TheSépharose® 4B-IDA gel was prepared according to the protocol describedby Sundberg and Porath (J. Chromatogr., 1974, 90, 87-98). TheNovarose®-IDA gel results from the same protocol as that described aboveat point 1 for the synthesis of the IMAdSEC gel, where only the secondand the third steps were carried out (no prior activation of the gelwith PEG). 2 ml of gel are applied to a column (diameter 1 cm) andlow-pressure chromatography (1 ml/min) is carried out. 10 ml ofultrafiltrate from a patient, whose β2-microglobulin concentration is 20μg/ml, are passed over each of the 3 different gels in closed circuitfor 20 minutes. The equilibration and the rinsing of each column afteradsorption of the ultrafiltrate are performed with an MMA buffer of pH7.0 (MMA=MOPS, MES, Acetate, 25 mM each). The elution is carried outwith a discontinuous decreasing pH gradient (buffer, 25 mM MMA, pH 6.0,then pH 5.0, then pH 4.0 and 25 mM glycine at pH 3.0), and then with asolution of EDTA (50 mM) to detach the copper. The protein content ismeasured during the chromatography by reading the optical density (λ=280nm) with a detector placed at the outlet of the column. The assay ofβ2-microglobulin is carried out by an immunological assay (rabbitpolyclonal antibody anti-human β2-microglobulin, Dako, Denmark) using anephelometry apparatus (Beckman, USA). The various fractions areanalyzed by SDS-PAGE electrophoresis, according to the protocoldescribed by Laemmli (Nature, 1970, 227, 680-685), and staining of theproteins with silver nitrate. After desalting and concentration, thefractions are analyzed by mass spectrometry (ESI-MS for “ElectroSprayIonisation Mass Spectrometry” technique), whose sensitivity, determiningthe mass to the nearest dalton, makes it possible to identify themolecules.

Results:

The chromatography on Sépharosee® 4B-IDA-copper gel (FIG. 3a) showsthat, while the β2-microglobulin has a high affinity for the chelatedcopper, its elution occurs in the same fractions as the albumin (FIG.4A). All the proteins of the ultrafiltrate are adsorbed onto the gel,which therefore exhibits no specificity for β2-microglobulin.

The chromatography on Novarose®-IDA-copper gel (FIG. 3B) also shows thatthis type of gel allows the adsorption of all the proteins of theultrafiltrate (FIG. 4B). Its capacity in relation to copper which islower than that of Sépharose®-4B-IDA results, on the other hand, inelutions of proteins during the discontinuous pH gradient, unlike theSépharose® 4B-IDA gel (FIG. 4B versus 4A). Like the latter, it does notoffer specificity for β2-microglobulin (FIG. 4B).

The chromatography on Novarose®-PEG/IDA-copper gel, on the other hand,allowed the adsorption of solely the β2-microglobulin of theultrafiltrate from the patient. Its elution takes place at pH 3.0 as twodistinct peaks (FIG. 4C).

In the three types of chromatography, the analyses by nephelometryconfirm the complete disappearance of β2-microglobulin from theultrafiltrate fraction passed over the 3 types of gel and its elutionfrom the column.

ESI-MS analysis shows that the chromatography on IMAdSEC gel makes itpossible to pass from a fraction consisting of a starting mixture:albumin+β2-microglobulin, to a fraction eluted at pH 3.0 which containsonly β2-microglobulin (FIG. 5A versus 5B).

These results show the affinity of β2-microglobulin for the ligand(chelated metal, here copper) and the specificity offered by themolecular sieving (coupling of PEG) of the IMAdSEC gel compared with theconventional IMAC gels.

4. Capacity of the IMAdSEC-copper gel for β2-microglobulin

Procedure:

50 ml of ultrafiltrate from a uremic patient, containing 350 μg ofβ2-microglobulin (that is a β2-microglobulin concentration of 7 μg/ml)circulates in closed circuit for 150 minutes on 0.65 ml of IMAdSEC gelunder the same chromatographic conditions as above (flow rate=1 ml/min).The elution is carried out directly at pH 4.0 (FIG. 6).

Results:

After 150 minutes, the β2-microglobulin concentration measured bynephelometry is 2.3 μg/ml, that is a remaining β2-microglobulin quantityof 115 μg. Consequently, 235 μg of β2-microglobulin were bound to the0.65 ml of gel, which corresponds to a binding capacity of theIMAdSEC-copper gel of 360 μg/ml. SDS-PAGE and ESI-MS analysis of thefractions was carried out as described above. The quantity ofβ2-microglobulin, eluted at pH 4.0, is about 180 μg instead of 235 μgexpected. The difference may be explained by the absence of measurementof the rinsing and EDTA fractions which are also likely to containβ2-microglobulin.

These results suggest that, taking into account these performances andthis specificity for β2-microglobulin, a column of 500 to 750 ml ofIMAdSEC-copper gel would make it possible to remove 250 mg ofβ2-microglobulin, a quantity which corresponds to 5 liters of blood at aβ2-microglobulin concentration of 50 mg/l.

EXAMPLE 2 Separation and Purification of β2-microglobulin by a DeviceComprising the Coupling of an Ultrafiltration Module and an IMAdSECColumn

Procedure

The assembly represented in FIG. 7 is used. The ultrafiltration module(1) used is composed of 100 Polysulfone hollow fibers drawn from acommercial ultrafiltration module model Fresenius F80.

50 ml of ultrafiltrate from a uremic patient (β2-microglobulinconcentration=7 μg/ml) are passed in a closed circuit for 3 hours on theultrafiltration/column of IMAdSEC gel (0.65 ml of IMAdSEC gel)minimodule assembly. The chromatography conditions are those of Example1, namely: buffer, 25 mM MMA, pH 6.0, then pH 5.0, then pH 4.0 and 25 mMglycine at pH 3.0, then 50 mM EDTA to elute the copper chelated on thegel.

After 3 hours, the β2-microglobulin concentration in the reservoir ismeasured by nephelometry.

Results

The concentration passes from 7 μg/ml of β2-microglobulin (that is astarting quantity of 350 μg) to about 1 μg/ml (50 μg of β2-microglobulinremaining). Consequently, about 300 μg of β2-microglobulin were bound tothe 0.65 ml of IMAdSEC gel, which corresponds to a binding capacity ofthe IMAdSEC gel for β2-microglobulin of 461 μg/ml.

ESI-MS (FIG. 8) and SDS-PAGE (FIG. 9) analysis of the fractions showthat the β2-microglobulin was adsorbed specifically by the IMAdSEC gel.It is eluted as two main fractions at pH 4.0 and pH 5.0.

These results suggest that the IMAdSEC gel could be useful for theseparation of biomolecules and their isoforms such as for example normalβ2-microglobulin and glycated β2-microglobulin.

What is claimed is:
 1. A device for removing biomolecules comprising anultrafiltration module optionally upstream and in series with a dialysismodule, wherein this device further comprises a column containing anadsorbent gel combining the properties of size exclusion and affinitychromatographies, said adsorbent gel consisting essentially of apolysaccharide matrix onto which is grafted a polymer coupled to anaffinity ligand and having an adjustable cut-off of between 2 kDa and 60kDa, said column being mounted branching from said ultrafiltrationmodule.
 2. The device according to claim 1, wherein the adsorbent gelconsists of a matrix based on an agarose derivative onto which isgrafted polyethylene glycol coupled to iminodiacetic acid itself coupledto copper(I) ions and having a cut-off of 20 kDa.
 3. The deviceaccording to claim 2, wherein the biomolecule is serum β2-microglobulin.4. The device according to claim 1, wherein the device is anextracorporeal dialysis system.
 5. A method for removing biomoleculesfrom blood, said method comprising passing said blood in a deviceaccording to claim
 1. 6. The method according to claim 5, wherein thedevice comprises an adsorbent gel consisting of a matrix based on anagarose derivative onto which is grafted polyethylene glycol coupled toiminodiacetic acid itself coupled to copper(I) ions and having a cut-offof 20 kDa.
 7. The method according to claim 5, wherein the biomoleculeis serum β2-microglobulin.
 8. The method according to claim 5, whereinthe device is an extracorporeal dialysis system.
 9. A device forseparating and purifying biomolecules comprising a column containing anadsorbent gel combining the properties of size exclusion and affinitychromatographies, said gel consisting essentially of a polysaccharidematrix onto which is grafted a polymer coupled to an affinity ligand andhaving an adjustable cut-off of between 2 kDa and 60 kDa, said columnbeing mounted branching from a ultrafiltration module.
 10. The deviceaccording to claim 9, wherein the adsorbent gel consists of a matrixbased on an agarose derivative onto which is grafted polyethylene glycolcoupled to iminodiacetic acid itself coupled to copper(I) ions andhaving a cut-off of 20 kDa.
 11. The device according to claim 10,wherein the biomolecule is serum β2-microglobulin.
 12. The deviceaccording to claim 9, wherein the device is an extracorporeal dialysissystem.